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RESULTS BIO
Achievements :
Main results :
Our project is based on aptamers which are single-stranded DNA able to recognize a specific target with a high affinity. In order to find potential aptamers, we choose 2 strains of bacteria, S. aureus and E. faecium and we performed 10 rounds of SELEX (Systematic Evolution of Ligands by EXponential enrichment) on both bacteria.
After these 10 rounds of SELEX, gel electrophoresis were made in order to verify that a high concentration of potential aptamers are still present.
Figure 1: Gel electrophoresis of 10 rounds of SELEX for E. faecium The gel is a 4% agarose one and the ladder used was a 50 bp ladder from Promega. The gel ran at 120 volts during 1 hour approximately.
We can observe that for the candidate aptamers selected against E. faecium we always had a single band throughout the rounds of SELEX (Figure 1). This gel demonstrates that a selection was applied on the initial DNA library because the bands are less brighter at the rounds 9 and 10 than the firsts rounds. This suggests that the most part of non-specific aptamers of E. faecium were removed during the washes made during the SELEX and that the specificity of the potential aptamers for E. faecium may have increased over the rounds.
Figure 2: Gel electrophoresis of 10 rounds of SELEX for S. aureus The gel is a 4% agarose one and the ladder used was a 50 bp ladder from Promega. The gel ran at 120 volts during 1 hour approximately.
As the previous one, the figure 2 demonstrates that the potential aptamers against S. aureus were selected because the bands loses their brightness over the rounds. However, on the round 9, we can notice 3 bands instead of one. It may be due to non specific amplification of DNA during the PCR. Despite this, it seems that these non specific amplifications were removed during the washes because they are not revealed during the round 10. So, we may think it will not interfere with the candidate aptamers.
Figure 3: Gel electrophoresis of the plasmid pET28:GFP at different step of the cloning experiment. The gel is a 1% agarose one and the ladder used was a 1 kbp ladder from Thermo Fischer. The gel ran at 120 volts during 1 hour approximately.
On the figure 3, we can observe the different steps of the cloning experiment. First of all, the plasmid pET28:GFP was digested using the restriction enzymes HindIII and BamHI. We can see a decrease on its size around 5 kbp which is a sign that the digestion went well. Then, the insert, either aptamers against S. aureus or those against E. faecium were ligated into the plasmid in order to create the biobrick BBa_K3176020
Sequencing
10 rounds of SELEX were performed in order to select potential aptamer candidates. These aptamers were cloned into pET28:GFP in order to be sequenced as bacterial clones. A total of 15 bacterial clones for E. faecium and 39 for S. aureus were sequenced. Unfortunately, the majority of the sequences obtained did not correspond to an aptamer sequence. Only two bacterial clones contained the expected 40 nucleotides of our initial library. These two sequences were found for E. faecium strain, none was found for S.aureus. Here is an histogram showing the sequence for one of these two aptamers.